Project Details
Projekt
Dynamics of Peroxisomal Protein Transport
Applicants
Professor Dr. Christian Eggeling; Dr. Harald Platta
Subject Area
Biochemistry
Term
from 2013 to 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 219314758
The aim of Project 4 is to use optimized microscopy and biochemical methods to expand the knowledge on membrane-associated events during the peroxisome import process. In the first part, we will use advanced optical microscopy methods to 1) visualize the relative distribution of proteins such as PEX5 and PEX14 at the peroxisomal membrane, 2) elucidate the diffusion- and interaction-properties of the PTS1-receptor PEX5 in the cytosol and at the peroxisomes, and 3) disclose molecular details of the dynamical association of peroxisomes to the cellular cytoskeleton. These experiments will be performed in fixed and living cells, with nanoscale spatial resolution, in relation to the import properties of the respective peroxisome, and after modifying the functionality of specific peroxisomal proteins. For this, we will improve super-resolution optical fluorescence microscopy methods such as STED and STED-FCS (as developed in the first funding period) for observing the peroxisomal protein import in cells, and will complement them with further observation methods. We will specifically optimize the three-dimensional observation of molecular organizations and dynamics at the peroxisomes, methods for cellular labeling of the involved proteins, and tools for analyzing the acquired data.In the second part of the project, the dynamics occurring during matrix protein import will be analyzed using biochemical tools. A focus will be on the PTS2-dependent import pathway. 1) We will verify the ubiquitination of the PTS2-co-receptor Pex21p, identify and characterize the responsible enzyme cascades, and analyze the functional consequences. 2) We will dissect the individual steps of the PTS2-cargo transporting PTS2-receptor module, which consists of Pex7p and the co-receptors Pex18p and Pex21p, and for which we will utilize a defined import/export system. 3) We will investigate the dynamic assembly and disassembly of the import machine in context of the function of the ubiquitin-conjugating enzyme Pex4p. Preliminary data show, that the interaction mechanism of the Pex4p/Pex22p-complex with the import pore is different from the interaction mode of the known components, like the docking-, RING- or AAA-complex. Moreover, preliminary data indicate that the presence of Pex4p controls the dynamic assembly of the AAA-complex to the import pore, for which we will identify the underlying mechanism using systematic complex isolations on different genetically modified strains. In the third part, we will apply a complementary approach, which combines microscopic and biochemical methods to study the PTS2-dependent import cycle and the ubiquitination-dependent assembly and disassembly of the import machine.
DFG Programme
Research Units
International Connection
United Kingdom
